Bottom Line:
Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles.The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d.We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

Affiliation: Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080, USA. pantano@utdallas.edu.

ABSTRACTThis work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

Figure 8: TEM micrographs of control HeLa cells that were incubated for 60 h at 37°C in DMEM/FBS (no DM-SWNTs). All slices were treated with uranyl acetate to stain membranes and lead citrate to stain the nuclear body. Colored arrows represent selected cell organelles: nuclei (red), mitochondria (green), Golgi bodies (yellow), vacuoles (blue), and the nucleolus (pink). Micrographs were normalized to the same grayscale as those in Figure 9.

Mentions:
TEM was used to examine the intracellular distribution of DM-SWNTs. Figures 8 and 9 show electron micrographs of HeLa cells incubated at 37°C for 60 h in DMEM/FBS (no SWNTs) or DM-SWNT dispersions, respectively. Colored arrows are used to denote the nucleolus and nucleus, vacuoles/vesicles, Golgi bodies, and mitochondria. In addition, it is important to note that all micrographs shown in Figures 8 and 9 were acquired from cells sliced in the plane of the nucleolus, as denoted by the low-magnification micrograph shown in Figure 8A. The first observation from the comparison of control and DM-SWNT treated cells was the lack of any SWNT-like structures visible in or associated with Golgi bodies (compare Figure 8C with 9E) and mitochondria (compare Figures 8D,E with 9B). The most striking observations between control (n = 8) and DM-SWNT treated (n = 10) cells was the appearance of dense black aggregated material in the cytoplasmic vacuoles of the DM-SWNT treated cells (Figures 9A–D) that was not observed in control cell vacuoles (Figures 8D–F). In the highest magnification view of these material-filled vacuoles (Figure 9D), the observed material displays black features with 5–20 nm diameters and apparent lengths of 50–300 nm, which is similar to the dimensions of CoMoCAT SWNTs in our dispersions. Such observations are consistent with those of Dai and co-workers who used confocal fluorescence microscopy to image the co-localization of SWNTs coated with a dye conjugate of avidin and the fluorescent endocytosis marker FM 4–64 [12,13].

Figure 8: TEM micrographs of control HeLa cells that were incubated for 60 h at 37°C in DMEM/FBS (no DM-SWNTs). All slices were treated with uranyl acetate to stain membranes and lead citrate to stain the nuclear body. Colored arrows represent selected cell organelles: nuclei (red), mitochondria (green), Golgi bodies (yellow), vacuoles (blue), and the nucleolus (pink). Micrographs were normalized to the same grayscale as those in Figure 9.

Mentions:
TEM was used to examine the intracellular distribution of DM-SWNTs. Figures 8 and 9 show electron micrographs of HeLa cells incubated at 37°C for 60 h in DMEM/FBS (no SWNTs) or DM-SWNT dispersions, respectively. Colored arrows are used to denote the nucleolus and nucleus, vacuoles/vesicles, Golgi bodies, and mitochondria. In addition, it is important to note that all micrographs shown in Figures 8 and 9 were acquired from cells sliced in the plane of the nucleolus, as denoted by the low-magnification micrograph shown in Figure 8A. The first observation from the comparison of control and DM-SWNT treated cells was the lack of any SWNT-like structures visible in or associated with Golgi bodies (compare Figure 8C with 9E) and mitochondria (compare Figures 8D,E with 9B). The most striking observations between control (n = 8) and DM-SWNT treated (n = 10) cells was the appearance of dense black aggregated material in the cytoplasmic vacuoles of the DM-SWNT treated cells (Figures 9A–D) that was not observed in control cell vacuoles (Figures 8D–F). In the highest magnification view of these material-filled vacuoles (Figure 9D), the observed material displays black features with 5–20 nm diameters and apparent lengths of 50–300 nm, which is similar to the dimensions of CoMoCAT SWNTs in our dispersions. Such observations are consistent with those of Dai and co-workers who used confocal fluorescence microscopy to image the co-localization of SWNTs coated with a dye conjugate of avidin and the fluorescent endocytosis marker FM 4–64 [12,13].

Bottom Line:
Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles.The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d.We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

Affiliation:
Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080, USA. pantano@utdallas.edu.

ABSTRACTThis work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.